Abstract Infections caused by the protozoan parasite Leishmania include cutaneous (CL), mucosal (ML), and visceral leishmaniasis (VL). The WHO classifies leishmaniasis as a neglected tropical disease with over 12 million current infections globally, and approximately 2 million new cases annually. Patients who recover from leishmaniasis develop protective immunity against reinfection, which altogether indicates that a vaccine is feasible. In the past, leishmanization, a process in which deliberate infections with a low dose of Leishmania major, etiologic agent of zoonotic cutaneous leishmaniasis (ZCL) causes a controlled skin lesion and provides > 90% protection against reinfection, was a common practice in ZCL-endemic regions. Under the current regulatory environment such practice is not acceptable due to possibility of complications including non-healing lesions. However, these studies suggest that live-attenuated parasites which don not cause a disease could be an effective vaccine for leishmaniasis. Genetically attenuated L. infantum and L. donovani including LdCen-/- have shown promise as a vaccine in animal models. However, using live-attenuated L. donovani as a vaccine in humans could raise safety concerns due to visceralizing potential of this Leishmania species. Attenuated dermatotrophic Leishmania that cross-protects against VL could be a safer vaccine against Leishmania because adverse events (e.g. development of a lesion as vaccination site) will be easy to monitor and can be treated using approved non-pharmacological interventions such as topical thermotherapy. Several clinical and preclinical animal studies have shown that an infection with dermatotrophic Leishmania such as L. major and L. tropica confers cross-protection against VL caused by L. donovani or L. infantum. Using CRISPR-Cas technology, we have generated antibiotic selection marker free centrin gene deficient L. major (LmCen-/-). Whole genome sequencing of LmCen-/- passed through mice multiple times has confirmed stable deletion of centrin gene without other mutations in the parasite genome. Our preliminary findings show that LmCen-/- are highly attenuated and fail to cause disease in immunocompromised animals. We have also found that immunization with LmCen-/- parasites induces disease protective Th1 response in hamsters and protects them against sand fly transmitted VL caused by L. donovani. Similarly, LmCen-/- immunization also protects against CL caused by L. major. These findings indicate that LmCen-/- is a promising vaccine for leishmaniasis. Our industry partner Gennova Biopharma has already established LmCen-/- production under cGMP conditions at their US-FDA approved facility. In this project, we propose to (Aim 1) perform phenotypic, genetic and biochemical characterization of GMP-LmCen-/-parasites and (Aim 2) determine whether GMP-LmCen-/- induce biomarkers of protection in peripheral blood mononuclear cells (PBMCs) from the patients with active VL, asymptomatics and those who have cured VL. The scientific promise of this project, if successful, could provide foundation for advancing LmCen-/- parasites as a vaccine against leishmaniasis in humans.